Type 2 diabetes is associated with obesity and elevated plasma levels of free fatty acids (FFA) and triacylglycerides (TAG). Obesity is also associated with the development of insulin resistance in insulin-sensitive tissues that results in hyperglycemia and hyperinsulinemia. b-cell failure in obesity-associated type 2 diabetes is believed to correlate with the intracellular accumulation of lipids that contribute to defects in insulin secretion and/or insulin and growth factor signaling necessary to maintain sufficient b-ceIl mass. The overall goal of this proposal is to define the cellular mechanisms whereby FFA and TAG exert their inhibitory effects on b-cell function in obesity-associated type 2 diabetes. Our recent studies have identified lipoprotein lipase (LpL) in b-cells, a key enzyme for catalyzing the hydrolysis of lipoprotein-associated TAG, to produce FFA for local cellular uptake. Our overall hypothesis is that LpL serves as a gatekeeper for the physiological import of FFA into b-cells analogous to that described for adipocytes. Furthermore, our new findings indicate that elevated concentrations of glucose and insulin enhance LpL enzyme activity in b-cells that may explain how b-cells continue to accumulate lipids in the setting of hyperglycemia and hyperinsulinemia associated with type 2 diabetes. In specific aim 1, we will: 1) further characterize the ability of nutrients, glucose and amino acids, and insulin to regulate LpL activity, expression and intracellular localization in rodent and human islets and b-cell lines, 2) assess the role of rapamycin, an inhibitor of mTOR, to regulate LpL and: lipid levels, and 3) evaluate LpL function in vivo. In specific aim 2, we will 1) examine the effects of enhanced FFA uptake by overexpression of FATP1 and ACS1, 2) characterize lipid droplet associated proteins, ADRP, perilipins and HSL, and 3) determine the regulation of lipid droplet synthesis and breakdown by phosphorylation and overexpression of lipid droplet associated proteins. Mitochondria exert a major role in glucose-stimulated insulin secretion, and mitochondrial activation is required for normal signal transduction. Recent studies suggest that FFA up-regulate mitochondrial uncoupling proteins (UCP) proposed to dissipate the proton gradient across the mitochondrial inner membrane. In specific aim 3, we will: 1) determine the role of UCP in mediating b-cell function by overexpressing UCP-2 in islets and b-cell lines, 2) assess the modulation of UCP-2 by increased levels of FFA in vitro and in vivo as described in specific aims 1 and 2. This experimental approach will be used to delineate the link between FFA and b-cell mitochondrial dysfunction.